[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro study was conducted with ERGP-IEM. The result of the study was:

ERGP-IEM was negative with and without metabolic activation when tested in the bacterial reverse mutation (Ames) assay according to OECD 471.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Company, lot # 6252020
- Purity, including information on contaminants, isomers, etc.: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: No information
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No information
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No information
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): solubolized in DMSO
- Preliminary purification step (if any): No information

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solubolized in DMSO

Target gene:

Tryptophan and Histidine operons.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No information

Test concentrations with justification for top dose:

33.3, 100, 333, 1000, 3333 and 5000 μg per plate per guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: compatibility with target cells

- Justification for percentage of solvent in the final culture medium:The test article formed a clear solution in DMSO at a concentration of approximately 200 mg/mL in the solubility test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.3x109 cells per milliliter
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No information
- Exposure duration/duration of treatment: 48 to 72 hours at 37±2°C.
- Harvest time after the end of treatment (sampling/recovery times): No information

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): No information
- Selection time (if incubation with a selective agent): No information
- Fixation time (start of exposure up to fixation or harvest of cells): No information
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: NA
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 0.3x109 cells per milliliter; The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
- Criteria for small (slow growing) and large (fast growing) colonies: No information

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: No information
- Any supplementary information relevant to cytotoxicity: Toxicity was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation

Rationale for test conditions:

Per OECD 471.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No information
- Data on osmolality: No information
- Possibility of evaporation from medium: No information
- Water solubility: No information
- Precipitation and time of the determination: Toxicity and degree of precipitation were scored relative to the vehicle control plate
- Definition of acceptable cells for analysis: No information

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the initial preliminary toxicity assay (A1), the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 3333 μg per plate with all conditions except tester strain WP2 uvrA in the presence of S9 activation. The S9 lot was not recorded for having been added to the S9 mix. There were no revertant counts for WP2A in the presence of S9 activation. An unacceptable vehicle control value was observed for tester strain TA100 in the presence of S9 activation (higher than the acceptable range stated in the protocol). Due to these observations, the preliminary toxicity assay was repeated with all conditions.
In the retest preliminary toxicity assay (A2), the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1000, 3333 or at 5000 μg per plate. Unacceptable vehicle control values were observed with tester strains TA100 and TA1537 in the presence of S9 activation (higher than the acceptable Historical Control Limit range stated in the protocol). As the preliminary toxicity assay is used to assess toxicity and precipitate to aid in the selection of dose levels for the mutagenicity evaluation, the unacceptable vehicle control values do not invalidate the assay. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Available

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No information
- Statistical analysis; p-value if any : No information

Ames test:
- Signs of toxicity : For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified. Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range. Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
- Individual plate counts : Available
- Mean number of revertant colonies per plate and standard deviation : No increase in revertant colonies was observed in treated plates

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : 0.3x109 cells/mL
o Number of cells plated in selective and non-selective medium : 0.3x109 cells/mL
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency : Available

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Contract research lab maintains records of historical positive control data for reference.
- Negative (solvent/vehicle) historical control data: Contract research lab maintains records of historical negative control data for reference.

Conclusions:

ERGP-IEM is not mutagenic in the presence or absence of metabolic activation up to 5000 μg/plate in the bacterial reverse mutation assay.

Executive summary:

ERGP-IEM was tested in a GLP-compliant, OECD 471 (1997) bacterial reverse mutation assay. Salmonella typhimurium TA98, TA1535, TA100, and TA1537 and Escherichia coli strain WP2uvrA were exposed to ERGP-IEM in DMSO at 33.3, 100, 333, 1000, 3333 and 5000 μg per plate with and without an exogenous metabolic activation system, Aroclor 1254-induced rat liver S9. No toxicity was observed. Positive and negative controls behaved as expected. Precipitate was observed beginning at 1000 or 3333 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based on the results of the study, ERGP-IEM is negative in the bacterial reverse mutation assay (Ames assay) in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the study, ERGP-IEM does not meet the criteria for classification according to GHS.